Bispecific antibodies are antibodies with dual epitope binding specificities, with one specificity being the capacity to bind a first epitope and a second specificity being the capacity to bind a second epitope.
Such bispecific antibodies are, in some embodiments, potentially valuable molecules for immunotherapy. For example, bispecific antibodies can crosslink cytotoxic effector cells to target cells (Segal and Snider, (1989) Chem. Immunol. 47:179), resulting in the killing of the target cell.
Although numerous bispecific antibodies have been shown effective in vitro (Gililand et al., (1988) Proc. Natl. Acad. Sci. USA 85:7719; Lanzavecchia and Scheidegger, (1987) Eur. J. Immunol. 17:105; Stearz and Bevan, (1986) Immunol. Today 7:241; Berg et al., (1991) Proc. Natl. Acad. Sci. USA 88:4732), few have been tested clinically as therapeutic agents. One of the reasons for the slow development of bispecific antibodies as therapeutic agents has been the difficulty in manufacturing them in sufficient purity and quantity.
Bispecific antibodies have been produced by chemical cross-linking, by hybrid-hybridomas (Milstein and Cuello, (1984) Immunol. Today 5:299) or transfectomas, or by disulfide exchange at the hinge of two different Fab'. The first method yields heterogeneous and ill-defined products. The second method requires extensive purification of the bispecific antibodies from many hybrid-antibody side products, the presence of which may interfere with the cell cross-linking activity. The disulfide exchange method applies essentially only to F(ab').sub.2, and is thus limited by the susceptibility of the monoclonal antibodies to cleavage by enzyme digestion (Parham, (1983) J. Immunol. 131:2895). Further, since Fab' have little affinity for each other, very high protein concentrations are required for the formation of the inter-Fab' disulfide bonds. The disulfide exchange method has been improved by the use of Ellman's reagent to modify one of the Fab' prior to oxidation with the other Fab', reducing the incidence of homodimerization (Brennan et al., (1985) Science 229:81). However, even with this improvement, heterodimeric F(ab').sub.2 can rarely be produced in better than 50% yield (Glennie et al., (1987) J. Immunol. 139:2367).
Thus, there remains a significant need for improved methods for efficiently producing bispecific antibodies and other similar compounds at high purity.